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The RAS Initiative

  • Posted: 09/22/2014

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RAS Pathways & Reagents

Frederick National Laboratory for Cancer Research (FNL) scientists have developed unique reagents to study RAS biology. As many of these as possible will be made available to the community of RAS researchers at nominal cost. Below are reagents now available for purchase.


KRAS Entry & RAS Pathway Clone Collections

Cost: US $38.48 (each requestor must pay shipping expenses) for each collection.

Contact: For additional information on the clone collections, to get more information on the Combinatorial Cloning Platform, or to order, please contact Dominic Esposito, Director, Protein Expression Laboratory, FNL at 301-846-7376 or dom.esposito@nih.gov.

For technical questions about the system, please contact Carissa Grose, Protein Expression Laboratory, FNL at 301-360-3427 or grosecl@mail.nih.gov.

Overview

The R3 KRAS Entry Clone Collection #1 is a set of vectors for use with the Gateway Cloning Platform (Life Technologies, Carlsbad, CA) to permit construction of KRAS expression vectors for in vitro and in vivo research.  While these clones can be used for standard Gateway cloning, their utility is greatly enhanced with the use of the FNL Combinatorial Cloning Platform (CCP), which allows simplified construction of vectors with different elements. Some uses of this system might be:

  • Construction of protein expression constructs with various fusion tags
  • Generation of expression constructs with different promoters for in vivo expression
  • Production of clones with fluorescent tags for localization experiments
  • Generation of constructs for making mutant cell lines or transgenic animals
  • Production of vectors for shRNA or miRNA delivery

The advantage of the CCP is in the exquisite specificity of the Multisite Gateway reactions, which permit linkage of multiple elements in a directional fashion and which involve no additional DNA amplification, thus ensuring the accuracy of the DNA sequence of the final products without the need for additional sequencing. There is also no need for restriction-based cloning processes which have a high rate of failure and may require optimization depending on the sites available in a given clone.

The KRAS Entry clone collection contains wild-type and mutant KRAS genes in two separate formats.  The “closed” format contains a Kozak translational initiation sequence and ATG start site at the 5’ end, and a stop codon at the 3’ end. These constructs can be used to make native or aminoterminally tagged constructs using Gateway vectors or the CCP. The “open” format has the same 5’ sequence, but lacks a stop codon and is in frame with the Gateway attB2 site at the 3’ end.  This allows production of C-terminal fusions using Gateway vectors or the CCP. These clones have been completely sequence validated and functionally tested in Gateway reactions to ensure proper performance.

A large collection of Gateway Multisite vectors tailored for mammalian expression experiments is available through Dr. Esposito. These plasmids combined with the KRAS entry clones allow mix–react–transform construction of plasmids with a variety of promoters (weak, strong, inducible, silencing-resistant) and N- and C-terminal fusions with fluorescent proteins, epitopes, and expression motifs.

Find more information at the PEL Combinatorial Cloning Platform

Gateway Recombinational Cloning

For more information on the Gateway system, please see Hartley JL, Temple GF, and Brasch MA. (2000) “DNA cloning using in vitro site-specific recombination.” Genome Res. 10, 1788-1795.

For more information on the FNLCR Combinatorial Cloning Platform (CCP), please see Wall VA, et. al. (2014). “Combinatorial assembly of clone libraries using site-specific recombination.” Meth. Mol. Biol. 1116, 193-208.


KRAS-4b wild-type and Mutant Expression Plasmids Clone Collection 1

KRAS Entry Clone Collection 1 (Wild-type KRAS and 10 point mutations / Open and close formats / Plasmid DNA and glycerol stocks of E. coli DH10B harboring each plasmid)

The NCI RAS Program collection of KRAS entry clones comprises 11 KRAS-4b genes, each in the closed (i.e., with a stop codon) and open (no stop codon, for C-terminal fusions) configuration. Both DNA and frozen E. coli cells are provided.  Miniprep plasmid DNA for all Entry clones is provided, and concentrations are noted on the vials.  In addition, glycerol stocks of E. coli DH10B cells containing the plasmids are provided. Gateway Entry clones are in the pDonr255 backbone and should be propagated with 50 μg/ml spectinomycin to ensure plasmid stability.  

The NCI KRAS entry clone collection includes wild-type KRAS-4b and the following mutants:

  • G12C
  • G12D
  • G12V
  • G13D
  • Q61L
  • Q61R
  • S181A
  • S181D
  • S181E
  • C185S

All clones are in a standard attL1-attL2 Gateway Entry vector (pDonr255) and contain a 5’ Kozak translation initiation sequence prior to the ATG start codon.

Clone IdentifierGeneC-terminus
R750-E01Hs.KRAS4bclosed
R750-E02Hs.KRAS4bopen
R750-E03Hs.KRAS4b G12Cclosed
R750-E04Hs.KRAS4b G12Copen
R750-E05Hs.KRAS4b G12Dclosed
R750-E06Hs.KRAS4b G12Dopen
R750-E07Hs.KRAS4b G12Vclosed
R750-E08Hs.KRAS4b G12Vopen
R750-E09Hs.KRAS4b G13Dclosed
R750-E10Hs.KRAS4b G13Dopen
R750-E11Hs.KRAS4b Q61Lclosed
R750-E12Hs.KRAS4b Q61Lopen
R750-E13Hs.KRAS4b Q61Rclosed
R750-E14Hs.KRAS4b Q61Ropen
R750-E15Hs.KRAS4b S181Aclosed
R750-E16Hs.KRAS4b S181Aopen
R750-E17Hs.KRAS4b S181Dclosed
R750-E18Hs.KRAS4b S181Dopen
R750-E19Hs.KRAS4b S181Eclosed
R750-E20Hs.KRAS4b S181Eopen
R750-E21Hs.KRAS4b C185Sclosed
R750-E22Hs.KRAS4b C185Sopen

 


Expression Plasmids for 15 RAS Pathway Genes

RAS Entry Clone Collection 1: 15 RAS pathway genes; Open and closed formats; Plasmid DNA and glycerol stocks of E. coli DH10B harboring each plasmid

Researchers within the NCI RAS Program have constructed a set of Gateway-compatible entry clones for the following 15 RAS pathway genes. All clones are available with and without stop codons (the latter configuration allows for C-terminal fusions). Miniprep plasmid DNA for all Entry clones is provided, and concentrations are noted on the vials.  In addition, glycerol stocks of E. coli DH10B cells containing the plasmids are provided. Gateway Entry clones are in the pDonr255 backbone and should be propagated with 50 μg/ml spectinomycin to ensure plasmid stability.  

Both DNA and frozen E. coli cells are provided. The genes include:

  • SOS1
  • SOS2
  • RASGRP1
  • RASGRP2
  • RASGRP3
  • RASGEF1A
  • KNDC1
  • RASA1
  • SYNGAP1
  • RASAL1
  • RASA4
  • BRAF 
  • RAF1
  • ARAF
  • PDE6D

All clones are in a standard attL1-attL2 Gateway Entry vector (pDonr255) and contain a 5’ Kozak translation initiation sequence prior to the ATG start codon. Genbank RefSeq identifiers are listed below to identify the specific isoforms of the genes in these clones.

Clone IdentifierGeneGenBank RefSeq IdentifierC-terminus
R702-E01Hs.SOS1NM_005633.3closed
R702-E02Hs.SOS1NM_005633.3open
R702-E03Hs.SOS2NM_006939.2closed
R702-E04Hs.SOS2NM_006939.2open
R702-E05Hs.RASGRP1NM_005739.3closed
R702-E06Hs.RASGRP1NM_005739.3open
R702-E07Hs.RASGRP2NM_153819.1closed
R702-E08Hs.RASGRP2NM_153819.1open
R702-E09Hs.RASGRP3NM_001139488.1closed
R702-E10Hs.RASGRP3NM_001139488.1open
R702-E11Hs.RASGEF1ANM_145313.2closed
R702-E12Hs.RASGEF1ANM_145313.2open
R702-E13Hs.KNDC1NM_152643.6closed
R702-E14Hs.KNDC1NM_152643.6open
R702-E15Hs.RASA1NM_002890.2closed
R702-E16Hs.RASA1NM_002890.2open
R702-E17Hs.SYNGAP1NM_006772.2closed
R702-E18Hs.SYNGAP1NM_006772.2open
R702-E19Hs.RASAL1NM_001193520.1closed
R702-E20Hs.RASAL1NM_001193520.1open
R702-E21Hs.RASA4NM_006989.5closed
R702-E22Hs.RASA4NM_006989.5open
R702-E23Hs.BRAFNM_004333.4closed
R702-E24Hs.BRAFNM_004333.4open
R702-E25Hs.RAF1NM_002880.3closed
R702-E26Hs.RAF1NM_002880.3open
R702-E27Hs.ARAFNM_001654.4closed
R702-E28Hs.ARAFNM_001654.4open
R702-E29Hs.PDE6DNM_002601.3closed
R702-E30Hs.PDE6DNM_002601.3open